GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes

• Published in: Plant and cell physiology 2008-11, Vol.49 (11), p.1699-1710
• Main Authors: Pelissier, H.C.(Washington State Univ., Pullman (USA)), Peters W.S, Collier, R, van Bel, A.J.E, Knoblauch, M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arabidopsis thaliana; Ca2+-dependent contractility; Calcium - metabolism; Composite plant; Contractile Proteins - genetics; Contractile Proteins - metabolism; EXPRESION GENICA; EXPRESSION DES GENES; Fabaceae; FLUORESCENCE; FLUORESCENCIA; Forisome; GENE; GENE EXPRESSION; GENES; Glycine max; Glycine max - genetics; Glycine max - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; LEGUMINOSAE; MEDICAGO TRUNCATULA; Medicago truncatula - genetics; Medicago truncatula - metabolism; Microscopy, Confocal; Molecular Sequence Data; PCR; Phloem-specific protein; Plant Leaves - genetics; Plant Leaves - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Promoter Regions, Genetic; PROTEINAS; PROTEINE; PROTEINS; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Regular Papers; Reverse Transcriptase Polymerase Chain Reaction; RNA, Plant - genetics; Sequence Alignment; Sequence Analysis, Protein; Sieve element occlusion (SEO) protein; Transformation, Genetic; VICIA FABA; Vicia faba - genetics; Vicia faba - metabolism; Vitis vinifera
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/pcp/pcn141

Forisomes are Casup(2+) driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Casup(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Casup(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.